Acetaminophen (APAP) hepatotoxicity is the most common cause of death due to acute liver failure in the developed world, and is increasingly recognized as a significant public health problem (1, 2). The initial event in APAP induced hepatotoxicity is a toxic-metabolic injury leading to hepatocyte death by necrosis and apoptosis. This results in secondary activation of the innate immune response involving up-regulation of inflammatory cytokines with activation of NK, NKT cells and neutrophils, which significantly contributes to hepatotoxicity and mortality (3, 4). The molecular pathways for innate immune activation after hepatocyte death are of great interest as they are likely a common pathway in sterile inflammation.
IL-1β is a very potent pro-inflammatory cytokine, and IL-1β levels are known to be increased during APAP hepatotoxicity (5, 6). In addition signaling through the IL-1 receptor (IL-1R) was recently shown to be important in APAP induced hepatotoxicity (7). The mechanisms by which cellular death results in up-regulation of IL-1β and activation of the sterile inflammatory response are not known. In contrast to sterile inflammation there is extensive data on IL-1β up-regulation by a variety of pathogens. Activation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) results in up-regulation of pro-IL-1β via a MyD88, NF-kB pathway. Analogous to other potent inflammatory steps, production of IL-1β requires a second signal resulting in caspase-1 mediated cleavage of pro-IL-1β to release the active molecule (8-10).
Our approach was to try to identify the two signals which were responsible for IL-1β production in APAP hepatotoxicity. TLR9 was of interest to us as a candidate molecule responsible for the first signal in sterile inflammation because in addition to being activated by bacterial DNA rich in un-methylated CpG motifs it can also be activated by DNA from mammalian cells (11) (12). When mammalian cells undergo apoptosis genomic DNA is modified by the caspase-activated DNAase (CAD)-mediated cleavage, and also aberrant methylation and oxidative damage (13-15). These apoptosis mediated changes increase the ability of mammalian DNA to activate TLR9 (16).
The activity of caspase-1 is regulated by a cytosolic protein complex called the inflammasome consisting of a NALP family member, the adaptor protein ASC and caspase-1 (17). A variety of molecules can result in activation of NALP pathways. These include molecules from dying mammalian cells causing activation of the inflammasome via NALP3, and molecules from gram negative organisms causing activation via IPAF (17). The NALP3 inflammasome was of interest to us as a candidate molecule responsible for providing the second signal required for IL-1β activity in APAP hepatotoxicity, and this was tested using mice deficient in caspase-1, ASC, or NALP3. We further aimed to identify clinically applicable strategies for down-regulating the caspase-1 inflammasome pathway, and test if they provide protection from APAP induced hepatotoxicity.